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THE    NEW   YORK    MEDICAL  JOURNAL,   JUNE   20,    1891. 

ILLUSTEATING   THE  AETICLE  BY   DE.   PEUDDEN   AND   DE.   HODENPYL. 

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Fig.  1. — Nodule  in  rabbit's  lung,  ten  days  after  intravenous  injection  of  dead  tubercle  bacilli.     Steamed  three  hours 


Fig.  2. — Nodule  in  rabbit's  lung,  twenty-eight  days  after 
intravenous  injection  of  dead  tubercle  bacilli.  Cult- 
ure steamed  two  hours. 


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Fig.  3.— New-formed  cells  in  capillaries  ot  the  rabbit's 
liver,  seventeen  days  after  intravenous  injection  of 
dead  tubercle  bacilli.     Culture  steamed  four  hours. 


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Fig.  4. — Diffuse  cell-proliferation  in  vessels  of  rabbit's  liver,  twenty-five  days  after  intra- 
venous injection  of  dead  tubercle  bacilli.     Culture  steamed  an  hour  and  a  half. 


Digitized  by  the  Internet  Archive 

in  2010  with  funding  from 

Boston  Library  Consortium  IVIember  Libraries 


http://www.archive.org/details/studiesonactionoOOprud 


THE   NEW  YORK   MEDICAL  JOURNAL,  JUNE  20,    1891. 


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Fig.  0. — Small  ^circumsoribed  epithelioid  cell-mass  in 
I'ahbit's  livei-,  surrouiided  by  zone  of  small  spberoidal 
cells,  twenty-five  days  after  intravenous  injection  of 
dead  tubercle  bacilli.  Culture  boiled  an  hour  and 
a  half. 


Fig.  5. — Small  circumscribed  area  of  cell-proliferation  in 
vessels  of  rabbit's  liver.     Same  animal  as  in  Fig.  i. 


Fir    8 — Small  Iner  nodule  composed  of  epithelioid 
cells.     Same  animal  as  m  Figs.  6  and  9. 


Fig.  7. — Small  nodule  in  rabbit's  liver  consis^ting  ot  a  mass  of 
epithelioid  cells,  forty-eight  days  after  intravenous  injec 
tion  of  dead  tubercle  bacilli.    Culture  steamed  thiee  hour'. 


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Fig.  9. — Kodule  in  liver  composed  of 
epithelioid  and  giant  cells.  Same 
animal  as  in  Figs.  7  and  8. 


Fig,  10. — Complex  nodule  in  rabbit's  liver,  consisting  of  a  congeries  of  epitheli- 
oid and  giant  cell  masses,  thirty-five  days  after  intravenous  injection  of  dead, 
tubercle  bacilli.    Culture  steamed  two  hours.    Very  few  bacilli  are  remaining 


STUDIES  OH"  THE 

ACTION  OF  DEAD  BACTERIA  IN 
THE  LIYING  BODY 


BY 

T.  MITCHELL  PRUDDEX,  M.  D. 

DIRECTOR 

AND  EUGENE   HODENPYL,   M.  D. 

FIRST  ASSISTANT  IN  PATHOLOGY  IN  THE    LABORATORY  OF  THE   AXUMNI 

ASSOCIATION  OF  THE  COLLEGE  OF  PHYSICIANS  AND  SURGEONS, 

NEW  YORK 


First  Papee. — Introductory 

Second  Paper. — A  Study  of  the  Tubercle  Bacillus 


REPEINTED  FROM 

THE  NEW   YORK   MEDICAL  JOURNAL 

FOR  JUNE  6  AND  20,  1891 


NEW     YORK 

D.    APPLE TON    AND    COMPANY 

1891 


Copyright,  1891, 
By  D.  APPLETON  AND  COMPATSIY. 


STUDIES   ON  THE  ACTION   OF 
DEAD    BACTEKIA  IN  THE  LIYING  BODY. 


FiEST  Article. — Inteodtjctoey. 

The  researches  of  the  past  decade,  bringing  to  light,  one 
after  another,  the  specidc  micro-organisms  of  some  of  the 
most  common  and  fatal  diseases,  have  been  so  surprising,  so 
definite,  so  fall  of  the  promise  of  fruitful  outlooks  upon 
hitherto  untrodden  fields,  that  we  have  scarcely  yet  had 
time  to  recover  from  the  glamour  of  the  new  light  or  to 
realize,  in  the  urgency  of  fresh  practical  problems,  the  exact 
extent  and  bearings  of  the  new  knowledge. 

For  a  time  it  seemed  enough,  and  even  more  than 
enough,  that  montli  by  month  the  proof  grew  stronger  that 
anthrax  and  tuberculosis  and  typhoid  and  erysipelas  and 
pneumonia  and  tetanus  and  diphtheria,  and  a  whole  group 
of  allied  "  wound  diseases  "  and  others  of  the  so-called  "  in- 
fections" class,  were  always  associated  with  certain  germs, 
each  peculiar  in  its  life  history,  and  each  standing,  as  we 
say,  in  an  setiological  relationship  to  its  particular  disease. 

But  as  we  have  little  by  little  become  accustomed  to  the 
new  light,  it  has  become  evident  not  only  that  we  are  still 
ignorant  about  the  relationship  of  micro-organisms  to  sev- 
eral extremely  frequent  and  important  infectious  diseases — 
the  exanthemata,  for  example — but  also  that  when  we  have 
learned  that  a  given  acute  infectious  disease  is  always  asso- 
ciated wiih  a  particular  form  of  germ,  when  the  life  history 
of  that  germ  is  made  out,  and  we  can  say  that  it  stands  in 
an  setiological  relationship  to  the  disease,  there  yet  remains 
a  series  of  accessory  problems  to  be  solved  in  each  particu- 


4  THE   ACTION   OF   DEAD   BACTERIA. 

lar  case  scarcely  less  important  than  the  establishinont  of 
the  invariable  association  of  the  germ  with  the  disease. 

We  are  just  beginning  fairly  to  realize  that  the  disease 
is  not  an  entity,  a  thing  imparted  by  the  invading  germ  to 
the  body,  but  that  it  is  the  result  of  the  reaction  of  the 
body  cells  in  the  presence  of  the  germs ;  that  the  body-cell 
factor  is  just  as  important  and  just  as  much  in  need  of 
studv  as  is  the  germ-cell  factor.  We  have  been  largely  for- 
getful hitherto,  as  with  painful  detail  the  characters  and 
preferences  and  metabolisms  and  vulnerabilities  of  the 
pathogenic  germs  have  one  by  one  been  brought  to  light, 
that  before  our  knowledge  of  the  acute  infectious  diseases 
can  be  at  all  complete,  the  characters  and  preferences  and 
metabolisms  and  vulnerabilities  of  the  body  cells  must  be 
subjected  to  an  equally  careful  scrutiny.  The  germ  side  of 
the  problem  is  new  and  fascinating;  the  man  side  is  old, 
and  cellular  pathology  is  a  phrase  familiar  to  our  ears.  But 
these  old  problems  have  become  fairly  new  in  their  new 
light,  and  can  not  too  soon  be  taken  up  afresh  if  our  knowl- 
edge of  the  acute  infectious  diseases  is  to  be  symmetrical 
and  of  lasting  use. 

Partly  by  clinical  observation  and  partly  by  laboratory 
studies  is  the  new  knowledge  of  the  man  side  of  this  theme 
to  be  acquired,  and  old  clinical  observations,  which  have 
lain  uninterpreted  or  misinterpreted,  and  new  facts  which 
the  new  points  of  view  can  not  fail  to  elicit,  will  surely  be 
fast  forthcoming. 

But,  returning  to  the  bacterial  side  of  the  problem,  it 
became  evident,  very  soon  after  the  definite  status  of  patho- 
genic germs  was  made  out,  that  something  more  than  their 
mere  presence  was  necessary  to  account  for  the  manifesta- 
tions of  the  acute  infectious  diseases. 

The  earlj  discovery  that  certain  pathogenic  germs  set 
free  poisonous  substances  of  one  kind  or  another  as  the 
result  of  their  life  processes,  and  the  evidence  that  these 
substances  were  directly  accountable  for  many  of  the  mani- 
festations of  the  acute  infectious  diseases,  drew  attention  to 
the  complexity  of  the  problems  involved,  called  in  the  serv- 
ices of  the  physiological  chemists,  and  for  a  time  it  seemed, 
and  still  does  to  many,  th;it,  after  all,  it  was  the  poisons 
which  the   bacteria  elaborated  and  sent  out  into  the  body 


THE   ACTION   OF   DEAD   BACTERIA.  5 

on  their  destructive  missions  which  was  the  most  important 
thing.  "Ptomaines"  became  a  favorite  word.  When  we 
had  said  that  a  given  germ  produced  a  given  disease  or  ef- 
fect by  the  elaboration  of  a  given  ptomaine,  it  seemed  to 
many  fairly  utireasonable  to  ask  for  any  further  explanation 
of  the  acute  infections  diseases.  The  germs  were  relegated 
to  the  more  humble  function  of  poison-factories,  and  the 
ptomaines  were  invested  with  the  insignia  of  malevolent 
power.  The  pendulum  seems  disposed  to  swing  back  germ- 
ward  now,  and  in  this  paper,  which  is  preliminary  to  the 
record  of  some  experimental  studies  made  by  Dr.  Iloden- 
pyl  and  the  writer,  an(i  shortly  to  follow,  on  the  tubercle 
bacillus,  it  is  my  purpose  briefly  to  review  a  series  of  recent 
studies  on  the  germ-cell  bodies  which  throw  a  curiously  in- 
teresting new  light  on  some  old  boilycell  problems. 

A  very  curious  vital  phenomenon  which  has  long  been 
known  in  certain  unicellular  organisms — such  as  the  fresh- 
water amoiba  and  in  the  leucocytes  of  both  the  cold  and 
warm-blooded  animals — is  their  response  by  movement 
to  contact  with  solid  substances.  Thus  the  amoeba  floating 
free  in  fluids  tends  to  assume  a  spheroidal  form  and  to  re- 
main immobile.  When,  however,  under  suitable  conditions, 
it  touches  a  solid  surface,  like  that  of  a  glass  slide,  it  sends 
out  pseudopodia  and  performs  those  curious  progressive 
evolutions  known  as  the  amoeboid  movement.  Essentially, 
the  same  series  of  movements  is  observed  in  leucocytes 
when  they,  under  favorable  conditions,  come  in  contact  with 
solid  surfaces — such  as  a  glass  slide  or  the  walls  of  the  body 
lymph  spaces.  This  faculty  in  these  primitive  forms  of 
life,  consisting  of  a  simple  lump  of  protoplasm,  is  called 
tactile  sensibility,  and  it  is  in  virtue  of  this  that  many  of  the 
remarkable  and  useful  evolutions  of  the  leucocytes  in  the 
body  transpire. 

It  was  found  by  Pfeflfer  (1),  a  good  while  ago,  that  some 
of  the  lowly  vegetable  organisms  endowed  with  locomotion 
— the  Flagellata,  Bacteria,  etc. — were  capable  of  moving 
toward  or  away  from  certain  substances  which  exerted  a 
chemical  action  upon  them.  This  property  he  designated 
as  chemotaxis,  and  further  postulated  &%  positive  chemotaxis 
the  attracting  effect,  and  as  negative  chemotaxis  the  repel- 
ling effect  on  such  organisms  of  the  chemical  substances. 


6         THE  ACTION  OF  DEAD  BACTERIA. 

Pfeffer  has  shown  that  mobile  bacteria  move  toward  nu- 
trient substances,  such  as  beef-tea,  and  Engelmann  and 
others  (2)  have  demonstrated  their  movement  toward  oxy- 
gen, both  effects  being  apparently  due  to  the  positive  chemo- 
tactic  action  of  tliese  substances.  Stahl  (3)  showed  that 
similar  properties  exist  in  the  plasmodia  of  myxomycetes. 
This  movement  has  been  proved  to  be  due,  not  to  currents 
in  the  fluids,  not  to  diffusion,  but  to  the  specific  action  of 
the  particular  chemical  substances  in  question  on  living  or- 
ganisms. 

Tbe  chemotactic  powers  of  the  juice  of  raw  potatoes, 
which  contains,  as  Pfeffer  showed,  potash  salts  and  aspara- 
gin,  has  been  used  in  capillary  tubes  by  Ali  Cohen  (4)  to 
separate  mobile  from  immobile  bacteria  in  mixtures.  In 
this  way  he  found  that  be  could  separate  cholera  and  ty- 
phoid bacilli  from  the  numerous  other  forms  in  fjeces,  and 
thus  make  easier  the  obtaining  of  pure  cultures  for  diagnos- 
tic purposes. 

Now,  the  same  condition  of  affairs  exists  in  the  leucocytes 
of  both  the  cold-blooded  and  warm-blooded  animals,  and 
the  conditions  and  bearings  in  them  of  this  positive  and 
negative  chemotaxis  wore  studied  in  detail  by  Massart  and 
Bordet(5)  and  by  Gabritchev&ki  (6)  in  1890.  Tbe  latter 
observer  has  grouped  as  the  result  of  his  experiments  cer- 
tain chemical  substances  in  accordance  with  their  action  in 
this  way  upon  leucocytes.  Tlius,  inthe  group  of  substances 
exciting  a  neixative — repelling — chemotaxis,  we  have  con- 
centrated salt  sohition,  10  per  cent. ;  lactic  acid  ;  quinine, 
0*5  percent.;  alcohol,  10  percent. ;  chloroform;  jetpiirity  ; 
glycerin  ;  bile.  Substances  having  no  effect — indifferent 
chemotaxis — are  distilled  vpater;  dilute  salt  solution,  0*1 
to  1  percent.;  carbolic  acid,  1-per-cent.  solution;  antipy- 
rine  ;  glycogen  ;  peptone  ;  beef-tea  ;  blood  ;  aqueous  hu- 
mor. Among  the  most  prominent  substances  exciting  a 
positive  chemotaxis  are  especially  sterilized  and  non-ster- 
ilized cultures  of  various  pathogenic  and  non-pathogenic 
bacteria. 

The  general  method  of  testing  the  powers  of  these  vari- 
ous substances  is  to  fill  small  capillary  glass  tubes,  closed 
at  one  end,  with  the  substance  to  be  tested,  and  to  thrust 
these  beneath  the  skin  of  an  animal.     After  a  few  hours 


THE   ACTION   OF   DEAD   BACTERIA.  7 

these  tubes  are  withdrawn  and  their  contents  examined. 
Into  tubes  filled  with  substances  inciting'  positive  chemo- 
taxis  the  leucocytes  crowd  in  great  numbers,  while  they  are 
held  away  from  tubes  having  negative  chemotactic  contents, 
and  when  filled  with  indifferent  substances  there  is  no  ef- 
fect at  all.  While  the  tactile  sensibility  of  leucocytes  may 
cause  them  to  cluster  in  small  numbers  about  the  surface  of 
the  glass  tubes,  the  effect  of  this  property  in  the  leucocytes 
is  altogether  insignificant  as  compared  with  the  chemical 
substance  exciting  positive  cheraotaxis. 

It  appears,  then,  that  there  are  certain  substances  asso- 
ciated with  bacteria  wliich  excite  in  the  leucocytes  a  move- 
ment toward  the  germs.  The  culture  medium  itself  has  no 
such  effect,  but  the  action  is  developed  equally  whether 
the  cultures  be  living  or  have  been  killed  by  boiling.  It 
would  thus  appear  that  either  the  bacteria  themselves  or 
some  result  of  their  life  and  growth  must  be  the  exciting 
agency. 

Under  the  dominant  views  regarding  the  significance  of 
the  various  chemical  substances  set  free  by  bacteria  as  the) 
grow,  it  has  been  assumed  that  it  was  largely  under  the  ex 
citing  influence  of  the  ptomaines  that  leucocytes  exhibited 
the  phenomena  of  chemotaxis  in  the  presence  of  bacteria, 
A  practical  bearing  was  given  to  the  subject,  under  the  in- 
fluence of  this  view,  by  the  assumption  that  in  the  process 
of  suppuration,  as  commonly  induced  by  various  species  of 
bacteria,  the  leucocytes  gathering  at  the  inflammatory  foci 
were  drawn  thither  in  virtue  of  their  chemotactic  properties 
which  the  metabolic  bacterial  poisons  brought  into  play.  To 
this  view  the  doctrine  of  phagocytosis,  as  held  by  Metsch- 
nikoff  and  his  adherents,  readily  attached  itself,  and  we  had 
a  well-rounded  hypothesis,  in  accordance  with  which  the 
leucocytes,  drawn,  in  virtue  of  their  chemotaxis,  into  the  vi- 
cinity of  invading  bacteria,  at  once  set  to  work  to  destroy 
them,  and  with  them  the  poison  sources  which  were  stimu- 
lating excessive  cell  inroads. 

But  while  these  observations  were  going  on,  an  allied 
but  quite  independent  series  of  experiments  was  being  car- 
ried out  by  Buchner  and  his  associates  in  Munich,  which 
have  thrown  a  new  and  apparently  most  significant  light 
upon  both  the  phenomena  of  chemotaxis  and  the  nature 


8         THE  ACTION  OF  DEAD  BACTERIA. 

of  snppnratioTi.  To  these  experiments  let  us  then  briefly 
turn. 

While  it  is  fully  established  that  a  true  suppurative  in- 
flammation may  be  experimentally  induced  by  a  variety  of 
inorganic  substances,  it  is  still  true  that  the  suppurative 
processes  which  occur  in  the  body,  either  as  independent 
lesions  or  as  complications  of  a  variety  of  diseases,  are  prac- 
tically always  due  to  the  action  of  bacteria.  So  that  in  a 
clinical  sense  the  summary  statement,  "  no  suppuration  with- 
out bacteria,"  is  true.  While,  as  above  indicated,  it  has 
been  the  general  belief  of  late  that  the  metabolic  products 
of  bacterial  life,  the  "ptomaines"  or  the  "toxines,"  were 
the  active  agents  in  inducing  suppuration,  this,  save  in  a 
few  instances,  has  not  been  proved. 

Biichner  (V),  in  the  course  of  some  experiments  on  the 
introduction  of  anthrax  spores  and  anthrax  bacilli  into  the 
trachea  of  rabbits  and  guinea-pigs,  had  observed  some  time 
ago  that  while  the  introduction  of  the  bacilli  was  followed 
by  an  intense  inflammatory  reaction  of  the  lung  tissue,  with 
accumulation  of  leucocytes,  fibrin,  etc.,  in  the  air  spaces,  the 
introduction  of  the  spores  alone  was  followed  by  no  such 
marked  inflammatory  reaction,  but  that  the  spores  entered 
the  blood  channels  and  induced,  in  due  time,  the  usual  sys- 
temic effects  of  anthrax  poisoning. 

There  is  one  factor — so  reasoned  Buchner — which  has 
not  been  taten  definitely  into  the  account  in  the  causation 
of  suppurative  inflammation  by  bacteria,  and  that  is  the 
possibility  that  the  effect  may  be  produced,  not  by  the 
ptomaines,  not  by  the  toxines  already  so  much  studied,  but 
by  the  albuminoid  constituents  of  the  bacterial  cells  them- 
selves. If  this  were  true,  then  the  inteuse  exudative  inflam- 
mation in  the  lungs  following  the  introduction  of  the  an- 
thrax bacilli  might  be  explained  by  the  local  disintegratiun 
of  the  bacilli  and  the  setting  free  of  their  potent  proteid 
constituents,  while  no  such  effect  would  follow  the  intro- 
duction of  spores. 

It  has  been  repeatedly  shown  by  numerous  observers  (8) 
that  sterilized  cultures  of  various  pyogenic  bacteria — such  as 
Staphylococcus  pyogenes  aureus,  Bacillus  pyocyaneus,  etc. — 
were  as  capable  of  producing  suppuration  as  were  the  fresh 
living  cultures.     But  it  was  believed  that  this  was  due  to 


THE   ACTION    OF   DEAD    BACTERIA.  9 

the  retention  of  a  toxic  substance  furnished  by  the  life  pro- 
cesses of  the  ^erm  which  had  not  been  destroyed  by  the 
sterilization,  but  clung"  about  the  dead  gern3  bodies.  Al- 
though Wyosokowitsch  had  filtered  off  the  fluid  from  steril- 
ized anthrax  cultures  and  found  that  the  filtrate  was  not 
pyogenic,  while  the  solid  material  was,  he  inferred  only 
that  the  toxic  material  assumed  to  cause  suppuration  was 
not  soluble  in  the  nutrient  fluid. 

Buchner  had  also  shown  (9),  in  the  course  of  some  other 
experiments,  that  the  sterilized  emulsion  of  the  so-called 
pneumo-bacillus  of  Friedlander,  subcutaneously  injected, 
could  cause  suppuration  in  rabbits  and  guinea-pigs.  He 
found  further  that  if  such  a  sterilized  emulsion  were  allowed 
to  stand  for  some  time,  so  that  the  solid  could  be  separated 
from  the  fluid  parts  of  the  mass,  the  fluid  part  did  not  cause 
suppuration,  while  the  solid  part  did.  That  the  effect  of 
such  sterilized  bacterial  emulsions  was  not  due  to  their  me- 
chanical effects  in  the  tissues  was  shown  by  such  control 
experiments  as  the  introduction  of  powdered  charcoal,  in- 
fusorial earth,  magnesia,  potato  emulsion,  etc.,  beneath  the 
skin,  with  negative  results. 

By  a  series  of  manipulations  similar  to  that  practiced 
with  the  pneumo-bacillus,  Buchner  now  tested  the  effect  of 
sterilized  emulsions  of  cultures  of  seventeen  different  species 
of  bacteria,  among  which  may  be  mentioned  Staph ijlococcus 
pyogenes  aureus,  Staphylococcus  cerens  flavus,  Sarcina  au- 
rantiaca,  Bacillus  prodigiosus.  Bacillus  fitzianus.  Bacillus 
cyanogenus,  Bacillus  megatherium.  Bacillus  suhtilis,  Bacillus 
coli  cominunis,  Bacillus  acidi  lactici,  Bacillus  anthracis,  Pro- 
teus vulgaris,  Finkler's  comma  bacillus,  etc.  The  injection 
of  one  cubic  centimetre  of  the  sterilized  emulsions  of  each 
of  these  germs  resulted  within  two  to  three  days  in  an 
aseptic — that  is,  bacteria-free — purulent  infiltration  in  the 
subcutaneous  tissue  at  the  seat  of  injection.  On  the  other 
hand,  the  clear  fluid  obtained  by  sedimentation  from  the 
sterilized  emulsions  of  Bacillus  cyanogenus.  Bacillus  mega- 
therium, and  Bacillus  anthracis,  induced  no  suppuration, 
while  the  separated  sediment  invariably  did. 

While  it  thus  seemed  probable  that  the  albuminous  ma- 
terial of  the  bacterial  cell  was  at  least  chiefly  concerned  in 
inducing  suppuration  on  the  injection  of  sterilized  emul- 


10  THE    ACTIOK   OF   DEAD   BACTEHIA. 

sions,  this  was  not  yet  fairly  proved.  Buchner  now  sought 
to  strengthen  the  evidence  by  a  very  ingenious  experiment. 
The  modern  technique  of  staining  bacteria  with  the  aniline 
dyes  depends,  as  is  well  known,  upon  the  power  of  these 
dyes  to  enter  into  chemical  combination  with  the  bacterial 
cell  plasma.  Acting  upon  this  principle,  Buchner  found 
that  if  he  added  to  a  sterilized  emulsion  of  the  pneumo- 
bacillus,  which  was  strongly  pyogenic,  an  aqueous  solution 
of  methvl  violet,  the  emulsion  was  wholly  bereft  of  its  pyo- 
genic powers.  Anent  of  this  interesting  bit  of  evidence  of 
the  importance  of  the  bacterial  cell  proteids,  Buchner  calls 
attention  to  its  bearing  upon  the  theory  of  the  antiseptic 
and  anlipyoDfenic  action  of  the  so-called  pyoktanin  of  Stil- 
ling, the  usefulness  of  which  in  practice  is  still  sub  judice. 

But  more  definite  proof  of  the  importance  of  the  bac- 
terid-protein  in  inducing  suppuration  was  still  needed,  and 
Buchner  proceeded  to  separate  it  from  cultures  of  the 
pneumo-bacillus  after  the  method  of  Necki,  by  digestion  of 
masses  of  culture  in  dilute  potash  and  precipitation  with 
acetic  or  hydrochloric  acid.  The  precipitate  separated  by 
filtration  was  again  dissolved  in  dilute  potash  sohition  and 
reprecipitated.  This  was  done  the  third  time,  and  at  last 
the  purified  product  was  brought  into  solution.  This  ma- 
terial gave  the  chemical  reaction  of  an  albuminoid  body. 
Subcutaneous  injection  of  this  material  in  rabbits  in  some 
cases  was  followed  by  a  gathering  of  leucocytes,  in  others 
not.  As  it  seemed  likely  that  on  simple  subcutaneous  in- 
jection the  material  was  readily  and  rapidly  absorbed  be- 
fore it  produced  local  effects,  recourse  was  had  to  a  method 
of  experiment  used  by  Councilman  in  his  well-known  studies 
on  suppuration  (10).  Small  glass  tubes,  drawn  out  at  the 
ends,  were  filled  with  the  pneumo- bacillus  protein,  sealed  up, 
and  steiilized  by  steam  for  an  hour.  These  were  then  in- 
troduced, with  strict  antiseptic  precautions,  beneath  the  skin 
of  rabbits,  shoved  away  from  the  opening,  and,  after  they 
were  healed  in,  their  tips  were  broken  off.  After  five  days 
the  tubes  were  exposed.  Around  the  openings  of  these,  as 
well  as  extending  deep  into  their  interior,  were  masses  and 
plugs  of  leucocytes.  Cultures  showed  no  living  bacteria. 
Control  experiments  with  tubes  filled  with  salt  solution 
showed  no  collection  of  leucocytes. 


THE   ACTION   OF   DEAD   BACTERIA.  H 

It  tlius  seemed  to  be  proved  that  the  pyogenic  action  of 
sterilized  cultures  of  Friedlander's  pneiimo-bacillus  is  due  to 
the  freed  albuminoid  constituents  of  the  bacterial  cell. 
That  such  a  freeing  of  tlie  contents  of  the  bacteiial  cells  oc- 
curs in  the  tissues  of  the  living;  body  seems  evident  from 
their  well-known  proneness  to  disintegration  and  the  devel- 
opment of  involution  forms  in  suppurative  foci. 

The  next  thing  to  be  done  was  to  carry  on  a  similar 
series  of  experiments  with  other  well-known  pathogenic 
bacteria.  To  this  task  Buchner  and  his  associates  addressed 
themselves  in  a  series  of  studies  as  yet  not  fidly  published 
(11).  But,  even  so  far  as  their  results  are  known,  some 
most  significant  facts  have  been  elicited.  Buchner  endeav- 
ored to  separate  by  the  method  of  Necki  (see  above)  the 
bacterio-protein  from  about  fifteen  species  of  bacteria,  but 
in  many  of  these  the  attempt  was  unsuccessful,  because 
sufficient  solution  and  extraction  of  the  proteid  ingredients 
of  the  germs  did  not  occur.  The  Bacillus  i^yocyaneus  gave 
the  most  abundant  albuminous  extract,  but  a  sufficient 
amount  was  obtained  from  Staphylococcus  pyogenes  aureus, 
Bacillus  typhosus,  Bacillus  suhtilis,  Bacillus  acidi  lactici, 
and  from  the  red  potato  ba':'illus  for  animal  expeiiment.  It 
was,  in  fact,  found  that  capillary  tubes  filled  with  the  puri- 
fied proteids  from  all  these  species  of  bacteria  and  placed 
beneath  the  skin  of  the  rabbit  showed  after  two  or  three 
days,  extending  into  the  open  end,  a  plug  of  fibrinous  pus 
several  millimetres  in  length.  This  plug  was  found,  on  mi- 
croscopical examination,  to  consist  largely  of  leucocytes. 

That  the  ordinary  chemical  decomposition  products  of 
bacterial  cell  life  are  not  concerned  in  inducing  this  posi- 
tive chemotaxis  in  the  leucocytes  was  shown  by  introducing 
beneath  the  skin  of  rabbits  tubes  filled  with  such  substances 
as  butyrate  and  valerianate  of  ammonia,  trimethylamin,  am- 
monia, glycocoll,  leucin,  tyrosin,  urea,  etc.  These  were,  for 
th"e  most  part,  wholly  without  effect  upon  the  leucocytes, 
only  glycocoll  and  leucin  exciting  in  some  cases  a  moderate 
chemotaxis,  not  at  all  to  be  compared,  however,  with  that 
of  the  bacterio-proteins. 

It  would  thus  seem  to  be  highly  probable,  if  not  abso- 
lutely proved,  that  the  power  of  exciting  positive  chemo- 
taxis, which  at   least  many   species  of  bacteria  display,  is 


12  THE   ACTION   OF   DEAD   BACTERIA. 

due  to  the  proteid  iuji-redients  of  their  bodies  when  these 
are  set  free,  as  they  may  be  naturally  when  the  germs  disin- 
tegrate in  the  tissues,  or  artificially  by  chemical  extraction. 

With  that  keenness  and  fertility  of  thought  which  char- 
acterizes Buchner's  work,  he  now  gave  wider  range  to  his 
investigation.  He  recognized  the  fact  that,  though  of  late 
the  phagocytic  action  of  the  leucocytes  has  been  especially 
emphasized  in  relation  to  bacteria,  this  is  by  no  means  their 
chiefest  or  most  constant  role.  Dispose  of  bacteria  the 
leucocytes  undoubtedly  do;  whether  after  themselves  killing 
them,  or  after  they  are  destroyed  by  other  ag;oncies,  ])as  not 
yet  been  fully  determined.  But  by  far  the  most  constant 
phagocytic  role  of  the  leucocytes  is  in  carrying  on  the 
process  of  resorption  and  disposal  of  useless  particles  and 
dead  material  in  the  living  body.  About  such  material 
they  gather  much  as  the}-  do  in  the  vicinity  of  bacteria, 
though  not  in  such  marked  degree  or  under  such  dramatic 
conditions. 

Now,  what  attracts  the  leucocytes  into  the  vicinity  of  a 
.particle  of  dead  and  useless  muscle,  or  cartilage,  or  connect- 
ive tissue  which  they  are  to  absorb  and  remove  ?  Certainly 
not  bacterial  poison,  certainly  not  bacterial  proteids,  for 
with  what  may  be  called  the  normal  phagocytic  functions 
of  the  leucocytes  bacteria  have  nothing  to  do.  Having 
shown  that  a  proteid  substance  derived  from  the  bacterial 
cells  was  capable  through  chemotaxis  of  attracting  leuco- 
cytes, Buchner  now  studied  in  a  similar  way  the  effects  of 
closely  allied  substances — namely,  the  so-called  vegetable 
caseins,  gluten  casein  from  wheat  and  legumin  from  peas, 
both  separated  by  precipitation  from  alkaline  solutions. 
Both  of  these  substances  were  capable  of  exciting  the  most 
marked  chemotaxis  in  the  leucocytes  of  rabbits.  More- 
over, as  it  has  been  shown  that  vegetable  casein  exists  as 
such  in  the  grain  of  cereals  and  of  the  leguminosse,  he  in- 
troduced beneath  the  skin  of  rabbits  or  guinea  pigs,  under 
strict  antiseptic  precautions,  masses  of  wheat  and  pea  meal, 
ami  found  that  within  two  days  these  masses  were  sur- 
rounded and  penetrated  by  enormous  masses  of  leucocytes. 
Cultures  from  these  masses  proved  the  entire  absence  of 
bacteria.  Starch  introduced  subcutaneously  under  the  same 
conditions  induced  no  gathering  of  leucocytes. 


THE    ACTION    OF   DEAD   BACTERIA.    /  I3  "Mf>^v 

That  this  gathering  of  leucocytes  was  due  to  cheraotaxis 
and  not  simply  mechanical,  owing  to  the  tactile  sensibility 
of  the  leucocytes,  was  shown  by  introducing  subcutaneouslv 
in  a  rabbit  in  one  place  an  emulsion  of  infusorial  earth  with 
0*7-per-cent.  salt  solution,  and  in  another  place  an  emulsion 
of  the  earth  with  glutin  casein.  In  the  first,  after  three  days, 
but  few  leucocytes  had  gathered  about  the  foreign  material, 
while  the  second  was  surrounded  and  partially  penetrated 
by  an  enormous  number  of  leucocytes. 

But  still  another  step  remained  to  be  taken.  As  the 
gathering  of  leucocytes  about  dead  organic  fragments  in  the 
tissues  which  are  to  be  removed,  as  so  often  happens,  can 
not  be  ordinarily  due  to  bacteria  or  bacterio-protein,  so, 
also,  interesting  as  the  observation  may  be,  can  vegetable 
proteins  have  no  part  in  the  matter.  So  alkali  albuminates 
were  prepared  and  purified,  in  a  manner  similar  to  that  em- 
ployed with  the  bacterial  and  other  vegetable  proteins  from 
muscle,  liver,  lungs,  and  kidney  of  rabbits.  These  tested 
in  the  same  way  were  all  found  to  strongly  attract  leuco- 
cytes when  introduced  beneath  the  skin  in  tubes.  Of  the 
alkali  albuminates  prepared  from  blood,  fibrin,  yolk  and 
white  of  egg,  only  the  blood  and  yolk  of  egg  showed  mod- 
erate power  of  exciting  positive  chemotaxis. 

These  experiments  show  that  it  is  only  ceitain  of 
the  decomposition  products  of  animal  tissue  which  pos- 
sess chemotactic  powers,  and  that  these,  as  a  rule,  are 
the  earlier  and  not  the  ultimate  products  of  the  decom- 
position. 

Finally,  as  it  has  been  shown  that  a  general  leucocytosis 
is  apt  to  be  associated  with  febrile  inflammatory  processes, 
Buchner  and  Roemer  studied  the  effects  of  intravenous  in- 
jections in  rabbits  of  these  various  chemotactic  proteids. 
They  found  that  within  eight  hours  of  their  introduction 
into  the  blood  there  was  a  marked  leucocytosis  lasting  for 
several  hours,  and  that  this  might  he  heightened  by  repeat- 
ed injections.  Thus  they  found,  by  a  daily  injection  of  2  c.  c. 
of  an  eight-per-cent.  solution  of  the  bacterio-jiroteins  of  Ba- 
cillus pyocyaneus,  the  relation  of  white  to  red  blood-cells, 
which  at  first  was  1  to  318,  was  on  the  second  day  1  to  126  ; 
on  the  third  day,  1  to  102  ;  on  the  fourth  morning,  1  to  73  ; 
and  on  the  same  evening,  1  to  38.     From  this  time  on  no 


14        THE  ACTION  OF  DEAD  BACTERIA. 

increase  was  noted.  The  absolute  number  of  the  red 
blood-cells  remained  unchanpted,  while  there  was  an  ab- 
solute sevenfold  increase  in  the  number  of  leucocytes. 
Gluten  casein,  as  well  as  alkali  albuminate  from  muscle, 
injected  into  the  blood,  showed  similar  but  less  pronounced 
effects. 

Considering  now  the  bearing-  of  all  these  experiments 
on  suppuration  and  on  physiological  resorption  of  dead  or- 
ganic materials  in  the  tissues,  it  would  appear  that  in  sim- 
ple resorption,  as  in  l>acterial  suppuration,  the  leucocAtes 
are  drawn  to  the  seat  of  operation  by  a  proteid  material. 
This  in  resorption  seems  to  be  furnished  by  the  dead  and 
disintegrating  ti^sues  themselves,  and  when  the  leucocytes 
have  gathered  up  a  certain  amount  of  refuse  in  their  bodies 
they  may  carry  it  away.  In  bacterial  suppuration,  on  the 
other  hand,  the  attracting  material  may  be  furnished  by  the 
protein  of  the  disintegrating  bodies  of  the  bacteria  them- 
selves, but  poisonous  ptomaines  fumished  by  the  live  bac- 
teria may  cause  the  destruction  and  degeneration  of  the  at- 
tracted leucocytes,  which  thus  collect  as  pus. 

Whether  the  ptomaines  themselves  may  not  indirectly 
furnish  chemotactic  material  by  causing  the  destruction  of 
the  tissue  elements  and  the  setting  free  of  their  albuAiinous 
constituents,  is  a  matter  requiring  further  study. 

It  is  also  not  improbable  that  the  limited  suppuration 
induced  by  bacteria-free  chemical  substances — such  as  tur- 
pentine, calomel,  etc. — may  be  due  to  the  chemotactic 
tissue-proteids  set  free  by  the  action  of  the  chemicals  on 
these  tissues. 

It  seems  probable  that  not  only  are  the  leucocytes  drawn 
toward  the  chemotactic  proteids  thus  produced  in  or  intro- 
duced into  the  body,  but  that  the  fixed  connective-tissue 
cells  are  stimulated  to  proliferation.  In  fact,  Buchner 
found  that  by  the  introduction  of  a  sterilized  emulsion  con- 
taining 3'5  milligrammes  of  pyocyaneus  protein  into  the 
forearm  of  one  of  his  associates,  a  severe  inflammation  was 
induced  with  all  the  symptoms  of  an  acute  typical  erysipe- 
las, with  lymphangcitis,  such  as  must  have  involved  the  fixed 
connective-tissue  cells.  On  the  fourth  day  the  inflamma- 
tory process  underwent  resolution.  Gluten  casein  induced 
similar  but  less  acute  effects. 


THE  ACTION  OF  DEAD  BACTERIA.        15 

These  most  clever  and  striking  researches  of  Bnchner 
would  seem  to  throw  niucli  light  on  the  whole  subject  of 
the  theory  of  suppuration,  and  to  promise  large  accessions 
to  ourknowleHge  of  inflammation  when  the  many  lines  of 
thought  and  study  which  they  suggest  shall  have  been  fol- 
lowed out. 

It  is  now  evident  that  an  aseptic  suppuration  is  possible 
under  a  variety  of  conditions. 

It  still  remains  true,  however,  for  the  purposes  cf  sur<>i- 
cal  practice,  that  the  suppurative  processes  as  we  see  them 
in  the  clinic  and  at  the  bedside  are  due  to  the  presence  of 
bacteria  of  one  form  or  another.  It  is  true,  also,  that  the 
suppurations  which  we  can  induce  experimentally  with  ster- 
ilized— that  is,  dead — bacterial  cultures,  or  with  certain  dead 
proteid  substances — aseptic  suppurations — are  limited  in 
their  duration,  extent,  and  destructive  power,  as  compared 
with  those  occurring  under  the  influence  of  living  germs. 
This  is  because  in  the  latter  case  the  growing  and  new 
forming  germs  may  keep  up  the  inflammation  once  alight 
to  an  almost  indefitiite  extent. 

We  purpose,  in  the  paper  which  is  to  follow,  to  detail 
some  results  of  a  series  of  experiments  on  the  action  of  dead 
tubercle  bacilli  on  the  living  tissue  not  less  striking  than  are 
those  which  show  the  power  of  other  sterilized  bacteria  to 
induce  suppuration. 

Bibliography. 

1.  Pfeffer.  Unters.  a.  d.  lotanischen  Institut  zu  Tubingen, 
1886-1888. 

2.  Engelmann.  Bot.  Zeitg.,  1881.  Prudden.  Medical  Rec- 
ord, March  26,  April  2,  1887. 

3.  Stall).   Zur  Biologie  der  Myxomjceten.  Bot.  Zeitg.,  1884:. 

4.  AH  Cohen.  Ctrlbl.  far  BaMeriologie,  Bd.  viii,  1890,  No. 
6,  p.  165. 

5.  Massart  and  Bordet.  Journal  public  par  la  soc.  royale 
des  sciences  medicates  et  natureltes  de  Bruxelles,  1890,  v. 

6.  Gabritchevski.    Annates  de  Vinst.  Pasteur.,  June  25,  1890. 

7.  Buehner.     Berl.  Min.  Woch.,  1890,  No.  30,  p.  673. 

8.  Bachner.     Op.  eit.,  p.  674. 

9.  Bachner.  .  Berl.  Uiii.  Woch.,  1890,  No.  10. 

10.  Councilman.     Virchow's  Archiv,  Bd.  92,  1883. 

11.  Buehner.     Berl.  Min.  Woch.,  1890,  No.  47. 


16  THE   ACTION    OF   DEAD   BACTERIA. 


Second  Aetiole. — The  AcTioisr  of  Steeilized  Cultures  of 

THE    TUBEECLE    BaCILLUS. 

We  have  seen  in  the  first  article  (1)  that  the  action  of 
bacteria  in  the  body  is  by  no  means  limited  to  the  chanyjes 
induced  by  those  substaoces  called  ptomaines  and  albu- 
moses,  or  toxaibarains,  which  are  set  free  by  living  germs. 
Tt  app?ars  that  the  proteid  constituents  of  the  bacterial 
cells  themselves,  when  these  are  set  free,  either  by  a  natural 
disintei^ration  of  the  germs  or  by  an  artificial  extraction, 
are  capable  not  only  of  stimulating  the  fixed  body  cells  to 
proliferative  changes,  but  may,  by  calling  into  play  the 
forces  involved  in  chemotaxis,  cau-^e  either  a  moderate  col- 
lection of  leucocytes  or  induce  marked  suppuration. 

It  is  probable  that  these  two  fairly  distinct  factors,  the 
eliminated  ptomaines  and  albumoses  on  the  one  hand,  and 
the  bacterio-proteins  on  the  other,  arc  both  involved  in 
many,  if  not  most,  of  the  acute  infectious  diseases.  But 
one  or  other  of  these  factors  is  apt  to  be  so  preponderant 
in  many  cases  as  to  fairly  dominate  both  the  symptoms  and 
lesions. 

For  example,  in  tetanus,  typhoid  fever,  and  Asiatic 
cholera,  the  toxic  substances  absorbed  into  the  body  at 
large  from  the  seat  of  growth  of  the  pathogenic  germs  may 
determine  profound  and  even  fatal  symptoms  without  much 
local  change  at  the  proliferating  germ  centers. 

On  the  other  hand,  in  many  of  the  local  suppurations — 
in  pneumania  and  in  tuberculosis,  for  example — the  most 
marked  effects  are  apt  to  be  induced  in  the  immediate 
vicinity  of  the  invading  germs. 

In  still  other  diseases — diphtheria,  for  example — we  may 
have  extreme  local  tissue  changes  together  with  profound 
systemic  effects. 

Finally,  under  a  variety  of  unusual  conditions,  patho- 
genic germs  which  commonly  induce  one  particular  order 
of  lesions  may  bring  about  most  striking  alterations  of 
another  class. 

As  an  example  of  this  variability  of  effect,  we  may  cite 


THE   ACTION   OF  DEAD   BACTERIA.  17 

the  suppurative  inflammations  which  not  infrequently  occur 
as  complications  of  typhoid  fever,  when  these  germs  lodge 
or  grow  in  unusual  situations. 

Perhaps  the  most  striking  example  of  a  pathogenic 
germ  whose  effects  are  closely  limited  to  the  immediate 
neighborhood  of  its  seat  of  growth  is  the  Bacillus  tuber- 
culosis. That  there  may  be  systemic  effects  induced  by 
the  absorption  into  the  body  at  large  of  poisons  eliminated 
by  the  tubercle  bacillus,  we,  of  course,  can  not  deny.  But 
if  such  there  be,  we  certainly  know  very  little  about  them, 
and  they  must  be  insignificant  in  comparison  with  the 
dominant  lesion  of  tuberculosis — namely,  the  production 
immediately  about  the  germs  of  a  new  short-lived  tissue 
having  a  moderately  characteristic  morphology  and  prone 
to  undergo  degenerative  changes  of  great  significance,  both 
to  the  germs  which  cause  it  and  to  the  integrity  of  the  in- 
vaded organ. 

It  is  because  of  the  peculiarly  direct  and  constant  re- 
lationship between  the  germ  and  the  lesions  which  it  in- 
duces that  we  have  selected  the  tubercle  bacillus  for  an 
experimental  stady  along  the  lines  which  our  preliminary 
paper  has  suggested. 

But,  before  detailing  our  own  experiments,  it  seems  de- 
sirable to  briefly  notice  some  observations  already  made  in 
this  field,  which  will  have  a  bearing  in  establishing  our 
point  of  view. 

A  number  of  studies  have  been  made  on  the  metabolic 
products  set  free  during  the  growth  of  the  tubercle  bacillus 
both  in  cultures  and  in  the  body.  But  these  studies  have 
been  made  from  such  a  variety  of  different  standpoints  and 
with  such  different  technic^ue  that  it  is  difficult  to  glean 
any  very  positive  and  definite  data  from  the  results. 

Hammerschlag  (2)  found  in  the  alcohol  and  ether  extract 
of  the  t'lbercle  bacilli,  fat,  lecithin,  and  a  poison  which  in- 
duced spasms  and  death  in  rabbits  and  guinea-pigs.  The 
residual  material  of  the  bacilli  contained  an  albuminoid  and 
cellulose.  He  could  not  separate  a  ptomaine  by  Brieger's 
method,  but  did  find  evidence  of  a  fever-causing  toxal- 
bumin. 

The  recent  publications  of  Koch  on  the  nature  and 
effects  of  substances  in  or  derived  from  cultures  of  the 
2 


.18  THE   ACTION   OF   DEAD   BACTERIA. 

tubercle  bai^illas  we  may  assume  to  be  familiar  to  all  our 
rea<lers.  But  the  manner  in  which  they  have  been  made 
known  and  the  lack  of  detail  in  the  description  of  the  mode 
of  preparation  of  his  "  lymph  "  leave  his  results  in  a  most 
unsatisfactory  state  of  indefiniteness,  so  far  as  the  scientific 
bearing  of  the  subject  is  concerned.  Some  substance  or 
substances,  it  would  appear,  either  set  free  during  the 
growth  of  the  tubercle  bacilli  or,  as  it  is  said,  extracted 
from  them  by  glycerin,  are  capable  of  exerting  rather  ill- 
understood  effects  upon  tubercle  tissue  in  the  body  and  are 
not  without  extreme  effects  in  many  cases  upon  the  body 
at  large.  With  the  therapeutic  bearing  of  the  substances 
contained  in  Koch's  tuberculin  we  have  here  nothing 
to  do. 

One  point,  however,  in  Koch's  last  publication  on  this 
subject  has  an  important  bearing  upon  the  subject  in  hand 
— namely,  the  statement  that  pure  cultures  of  the  tubercle 
bacillus  killed  by  boiling,  or  in  other  -ways,  are  capable  of 
inducing  local  suppuration  witb  no  other  effects,  if  intro- 
duced subcutaneotisly  into  the  guinea-pig. 

Hueppe  and  Scholl  (3)  cultivated  the  tubercle  bacilli  in 
a  glycerin-peptone  bouillon,  and  found  that  the  fluid  con- 
tained substances  having  essentially  the  same  eff"ects  upon 
animals  as  those  noted  by  Koch.  As  the  result  of  an  inter- 
esting series  of  experiments,  these  observers  came  to  the 
conclusion  that  the  specific  poison  of  Koch's  "  lymph"  did 
not  belong  to  the  proteins,  but  to  some  substance  or  sub- 
stances eliminated  by  the  bacilli  during  their  growth  and 
contained  in  the  culture  fluids,  and  which  were  not,  as  Kocb 
asserted,  extracted  from  the  culture  by  glycerin.  Ilucppe 
and  Scholl  separated  from  the  "  lymph  ''  which  they  had 
prepared,  and  which  seemed  to  be  practically  identical  with 
the  ''lymph"  of  Koch,  by  precipitation  with  alcohol,  a  sub- 
stance which  appears  to  contain  the  active  ingredient  of  the 
"  lymph."  They  separated  in  the  same  way  the  same  sub- 
stances from  Koch's  "lymph,"  and,  on  testing  these  in  tubes 
beneath  the  skin  of  animals,  they  foinid  that  in  both  cases 
this  substance  was  markedly  chemotactic. 

That  Koch's  '"  lymph  "  is  not,  as  it  is  furnished  in  the 
crude  condition,  chemotactic  and  does  not  tend  to  cause  ab- 
scesses,  may  be  due  to  the  fact  that  it  contains  glycerin, 


THE   ACTION   OF   DEAD   BACTERIA.  19 

which  exerts  an  influence  on  the  leucocytes  opposite  to  that 
which  positive  cbem-itactic  substances  display. 

Trudeau  (4),  working  also  with  fluid  cultures,  obtained 
materials  apparently  the  result  of  metabolic  processes  of 
the  tubercle  bacilli  which  induced  toxic  effects  upon  ani- 
mals. But  these  were  not  marked,  since  he  did  not  succeed 
in  sufficiently  concentrating  the  fluid  containing  the  mate- 
rials sought  for. 

Zulzer  (5)  extracted  from  agar  cultures  a  substance  which, 
on  purification  and  injection  subcutaneously  in  small  quan- 
tities into  rabbits  and  guinea-pigs,  caused  temporary  in- 
creased rapidity  of  respiration  and  an  elevation  of  tempera- 
ture.    Larger  does  were  fatal. 

Crookshank  (6)  separated  the  bacilli  from  glycerin-broth 
cultures  by  filtration,  and  concentrated  the  filtrate  by  evapo- 
ration. This  concentrated  filtrate,  injected  subcutaneously 
into  guinea-pigs,  induced  muscular  spasms,  a  lowering  of 
the  temperature,  and  death. 

Weyl  (7)  extracted  tubercle  bacilli  with  dilute  caustic 
soda,  and  found  in  this  extract  a  substance  which  seemed 
to  him  to  belong  in  the  mucin  group.  Subcutaneous  in- 
jection of  a  solution  of  this  substance  produced  in  animals 
a  local  necrosis  at  the  seat  of  injection. 

Maffucci  (8)  found  that  sterilized  cultures  of  tubercle 
bacilli  introduced  beneath  the  skin  of  guinea-pigs  in  con- 
siderable amounts  were  capable  of  inducing  a  chronic  poi- 
soning of  the  organism,  ending  in  death  from  marasmus  and 
great  destruction  of  the  red  blood-cells  in  the  spleen,  but 
does  not  note  any  marked  local  changes  beyond  the  pro- 
ducing of  an  abscess. 

Wyssokowicz  (9)  killed  the  tubercle  bacilli  in  cultures 
by  boiling,  and  these  on  subcutaneous  injection  in  a  rat 
caused  a  small  local  abscess,  containing  well-formed  and 
readily  stained  though  dead  tubercle  bacilli. 

An  emulsion  of  a  boiled  culture  of  the  tubercle  bacilli 
was  injected  into  the  abdominal  cavity  of  a  rat.  The  ani- 
mal was  killed  on  the  thirtieth  day.  A  few  small  nodules 
were  found  beneath  the  liver  which  consisted  of  a  central 
pus-like  mass  surrounded  by  a  thin  new-formed  tissue 
wall.  The  central  portion  of  these  nodules  consisted  of  tu- 
bercle bacilli  and  leucocytes,  while  their  walls  were  com- 


20  THE    ACTION   OF   DEAD   BACTERIA. 

posed  largely  of  endothelial  cells,  with  here  and  there  giant 
'cells  among  them.  Readily  stained  tubercle  bacilli  were 
scattered  through  the  walls  of  these  nodules,  and  the  epi- 
thelioid and  giant  cells  were  often  grouped  about  them. 
The  microscopical  examination  of  the  liver  revealed  numer- 
ous small  spheroidal  nodules  composed  of  epithelioid  cells 
and  leucocytes,  among  which  were  scattered  a  few  tubercle 
bacilli. 

We  know  that  various  kinds  of  substances,  organic  and 
inorganic,  when  introduced  into  the  body,  can  become  sur- 
rounded by  a  tissue  in  many  respects  resembling  tubercle 
tissue.  This  was  abundantly  shown  in  the  years  just  pre- 
ceding the  discovery  of  the  tubercle  bacillus  and  the  final 
establishment  of  tuberculosis  as  an  acute  infectious  disease. 
Whether  the  dead  tubercle  bacilli  in  these  experiments  of 
Wyssokowicz's  act  in  some  special  way  in  stimulating  the 
tissues  to  these  tubercle-like  growths,  or  whether  they  do 
this  simply  as  foreign  bodies,  must  be  shown  by  further 
studies. 

The  action  of  the  living  tubercle  bacillus  in  inducing 
the  local  lesions  of  tuberculosis  seems  to  be  quite  complex. 
But  there  appear  to  be  several  tolerably  distinct  classes  of 
influences  at  work. 

In  the  first  place,  there  seems  to  be  an  irritation  or 
stimulation  of  the  tissues  near  the  bacilli  which  results  in 
the  proliferation  of  cells,  primarily  and  notably  of  the  con- 
nective tissue  cells.  The  eJBFect  of  this  is  to  produce  what 
is  called  the  epithelioid  cell  tissue.  Following  this,  or  asso- 
ciated with  it,  there  is  a  local  action  either  on  the  leuco- 
cytes or  the  blood-vessels,  or  both,  which  leads  to  the  ac. 
cumulation  of  small  spheroidal  cells  either  in  the  periphery 
of  the  tubercle  or  distributed  through  it.  Generally  there 
is  a  necrotizing  action  which  results  in  the  coagulation 
necrosis  or  cheesy  degeneration  of  the  new-formed  tissue 
or  other  tissue  near  the  tubercle. 

The  giant  cells  which  form  such  striking  features  in 
many  phases  of  tuberculosis  appear  to  be  formed  under  the 
influence,  on  the  one  hand,  of  those  factors  which  induce 
cell  proliferation,  and,  on  the  other,  of  those  which  retard 
the  completion  of  the  cell  proliferation  when  once  it  is 
under  way.     The  impulse  to  cell  division  under  these  cir- 


THE   ACTION   OF   DEAD   BACTERIA.  21 

cumstances  is  in  so  far  successful  that  a  protoplasmic 
mass  is  formed  with  many  nuclei,  but  the  cousumraa- 
tion  of  this  attempt,  the  division  of  the  cell  bodies,  is  in 
abeyance. 

In  view  of  the  new  facts  regarding  the  singular  power 
of  the  bacterio-protcin  and  chemotaxis  which  the  researches 
briefly  summarized  in  our  first  article  have  revealed,  and 
the  promising  but,  it  must  be  confessed,  rather  obscure 
light  which  the  recent  studies  on  the  poisons  of  the  tu- 
bercle bacillus  have  elicited,  it  has  seemed  to  us  that  some 
further  studies  on  the  tubercle  bacillus  should  be  made 
with  the  view  of  learning,  if  possible,  in  more  detail  some- 
thing of  the  factors  which  are  concerned  in  developing  the 
complex  structures  which  we  call  tubercles  and  tubercle 
tissue. 

Our  first  series  of  studies  was  made  with  the  view  of 
determining  what  effects,  if  any,  are  produced  in  the  body 
of  the  rabbit  by  the  introduction  of  cultures  of  the  tubercle 
bacilli  killed  by  prolonged  boiling  and  freed  from  any  ma- 
terial elaborated  by  them  during  their  growth  on  the  artifi- 
cial culture  media,  so  far  as  such  substances  are  soluble  in 
the  culture  media,  or  in  water,  or  in  fifty-per-cent.  glycerin. 
That  is  to  say,  we  wished  in  these  experiments  to  learn 
whether  the  dead  bodies  of  the  tubercle  bacilli  alone,  apart 
from  any  of  their  elimination  or  soluble  metabolic  products, 
produced  changes  in  the  body  cells  of  living  rabbits,  and, 
if  so,  of  what  kind. 

Technique. — We  cultivated  the  tubercle  bacillus  for  this 
purpose  on  peptone  agar  with  six-per-cent.  glycerin,  and  in 
glycerin-peptone  bouillon.  In  all  cases  we  continued  the 
growth  until  it  became  voluminous,  and  we  obtained  iden- 
tical results  whether  the  cultures  were  two  months  or  six 
months  old,  and  whether  they  were  made  in  bouillon  or  on 
agar.  We  have  used  cultures  of  tubercle  bacilli  derived 
from  three  different  sources. 

The  solid  masses  of  culture  were  scraped  carefully  off 
from  the  agar,  or  filtered  from  the  culture-bouillon.  In 
some  cases  we  were  not  particular  to  remove  from  the  cult- 
ures what  culture-bouillon  clung  to  the  germ  masses.  But 
in  most  of  the  experiments  the  masses  of  bacilli  were 
washed  with  sterilized  distilled  water,  and  then  boiled  in  a 


22  THE   ACTION    OF   DEAD   BACTERIA. 

small  amount  of  sterilized  water  for  from  an  hour  and  a 
half  to  four  hours.  In  still  another  set  of  experiments  we 
first  carefully  washed  the  culture  masses,  and  then  boiled 
them  for  from  two  to  four  hours  in  tifty-per-cent.  glycerin 
with  water,  and  then  filtered  off  the  fluid,  washing  the 
germ  masses  before  making  an  emulsion  with  pure  water 
for  injection. 

We  have  found  that  the  condensation  fluid  in  glycerin- 
agar  cultures  and  also  the  glycerin  bouillon  in  which  the 
tubercle  bacilli  had  grown,  when  dropped  into  strong  alco- 
hol, gives  a  white  precipitate,  which,  according  to  the  studies 
of  Koch  (10)  and  Hueppe  and  SchoU  (11),  contains  the  act- 
ive principles  of  Koch's  tuberculin.  We  furthermore  found 
that  after  the  glycerin-agar  condensation  water  or  the  gly- 
cerin bouillon  in  which  the  tubercle  bacilli  are  grown  has 
been  carefully  washed  out  of  the  culture  masses  of  bacilli, 
no  more  of  this  tuberculin-containing  material  is  extracted 
from  the  bacilli  by  either  water  or  fifty-per-cent,  glycerin, 
even  after  boiling  for  four  hours.  It  would  thus  appear 
that  the  active  substance  in  Koch's  "lymph  "is  a  mate- 
rial elaborated  by  the  life  processes  of  these  germs  and 
set  free  in  the  culture  media,  as  shown  by  Hueppe  and 
Scholl  (3). 

After  steaming  the  bacilli  for  from  an  hour  and  a  half 
to  four  hours  in  the  steam  sterilizer,  and  separating  them 
from  their  metabolic  products  in  the  manner  described,  we 
have  made  an  emulsion  of  the  bacilli  in  sterilized  water 
which  we  have  used  in  our  experiments.  In  a  few  cases  we 
have  boiled  the  bacilli  together  with  a  small  amount  of  the 
culture  fluids  containing  their  metabolic  products,  but  the 
results  of  these  experiments  have  been  the  same  as  when 
we  have  carefully  separated  them. 

We  find  that  after  prolonged  boiling,  either  in  water  or 
in  fifty-per-cent.  glycerin,  while  many  of  the  bacilli  present 
the  usual  bizarre  broken  granular  involution  and  degenera- 
tion forms  so  often  described,  most  of  them  preserve  their 
form  intact,  and  are  stained  in  the  usual  way  by  the  Koch- 
Ehrlich  method.  The  broken  granular  forms  are  readily 
stained. 

We  have,  by  a  series  of  separate  experiments,  proved 
that  the  tubercle  bacilli  are  certainly  killed  by  the  pro- 


THE    ACTION   OF   DEAD   BACTERIA.  23 

longed  steaming  to  which  they  have  been  subjected,  of  from 
an  hour  and  a  half  to  four  hours. 

As  this  is,  of  course,  a  point  of  vital  importance  in  our 
experiments,  we  have  been  very  careful  about  this  prelimi- 
nary sterilizing.  We  have  used  the  Arnold  steam  sterilizer, 
which  is  one  of  the  most  efficient  forms  of  laboratory  ster- 
ilizers known  to  us.  The  material  to  be  killed  has  been 
invariably  placed  in  small,  thin-walled  flasks,  exposed  to 
the  full  action  of  live  steam.  The  duration  of  the  steam- 
ing was,  of  course,  carefully  reckoned  from  the  time  when 
the  chamber  of  the  steamer  and  its  contents  was  at  the  full 
boiling  temperature.  While  we  have  found  in  a  series  of 
preliminary  experiments  that  an  hour  and  a  half's  steaming 
sufficed  to  kill  the  cultures,  as,  from  what  we  know  of  the 
vulnerability  of  these  germs,  it  should,  in  all  except  one 
set  of  experiments,  we  continued  the  steaming  from  two 
to  four  hours,  the  average  time  being  about  two  hours  and 
a  half. 

The  masses  of  tubercle  bacilli  thus  steamed  have  been 
proved  to  be  dead,  first,  by  their  failure  to  grow  on  favorable 
culture  media;  and,  second,  by  the  much  more  delicate  test 
of  repeated  inoculations  in  guinea-pigs,  which  invariably 
gave  negative  results  so  far  as  the  development  of  tuber- 
culosis is  concerned.  Finally,  we  have  inoculated  guinea- 
pigs  with  materials  from  the  lesions  about  to  be  described 
as  induced  in  rabbits  by  the  dead  cultures,  and  obtained  in 
the  guinea-pig  only  negative  results. 

In  these  ways  we  have  satisfied  ourselves  that  the  ma- 
terial which  we  were  using  contained  in  every  case  no  liv- 
ing tubercle  bacilli,  but  only  their  dead  bodies. 

Experiments.  —  Subcutaneous  Injections.  —  We  first 
made  a  series  of  injections  of  the  emulsion  of  the  sterilized 
tubercle  bacilli  in  rabbits  beneath  the  skin  and  found  that 
in  a  considerable  proportion  of  cases  a  small  local  abscess 
is  developed  in  from  two  to  six  weeks.  This  abscess,  some- 
times firmly  encapsulated  with  fibrous  tissue,  contains  pus 
cells,  granular  detritus,  and  many  readily  stained  and  well- 
formed  tubercle  bacilli,  together  with  involution  forms. 
This  pus,  as  above  indicated,  on  inoculation  into  that  most 
susceptible  animal,  the  guinea-pig,  gave  negative  results ; 
no  tuberculosis  developed,  which  was  to  be  expected  if  the 


2i  THE    ACTION    OF   DEAD   BACTERIA. 

cultures  of  the  tubercle  bacilli  used  for  inoculation  had  been 
readily  sterilized.  We  have  further  made  cultures  of  this 
pus  with  a  view  of  determining  whether  this  suppuration 
might  not  be  due  to  some  of  the  ordinary  pyogenic  bac- 
teria, but  the  results  were  negative.  We  thus  find,  in  con- 
sonance with  the  results  of  Koch,  and  of  Hueppe  and 
SchoU,  that  dead  tubercle  bacilli  are,  under  certain  condi- 
tions, markedly  pyogenic. 

We  have  tested  the  chemotactic  powers  of  sterilized 
cultures  of  the  tubercle  bacillus  introduced  in  tubes  beneath 
the  skin  of  rabbits,  in  the  manner  described  in  our  first 
article,  and  found  in  every  case  after  six  days  a  plug  of 
leucocytes  extending  deep  into  the  broken  end  of  the 
tube.  We  have  introduced  the  dead  cultures  beneath 
the  skin  of  rabbits  inclosed  between  the  laminae  of  Zieg- 
ler's  plates.  Tn  from  three  to  five  days  the  capillary 
space  was  found  crammed  with  leucocytes.  The  dead 
tubercle  bacillus  thus  possesses  marked  positive  chemotac- 
tic powers. 

Peritoneal  and  Pleural  Injections. — We  next  made 
injections  of  the  emulsion  of  dead  tubercle  bacilli  in  con- 
siderable quantities  (2  to  3  c.  c.  of  a  milky  emulsion) 
into  the  peritoneal  and  pleural  cavities — in  four  animals 
into  the  peritoneal  cavity  ;  in  two,  into  the  pleural  cavity. 
We  obtained  one  positive  result  in  each  set  of  injections. 
The  others  were  all  negative. 

In  the  positive  cases,  whose  results  were  similar  in  both 
the  pleura  and  peritonaeum,  we  found  several  larger  and 
smaller  white  nodules  adherent  to  the  serous  surfaces. 
These  consisted  of  a  central  soft  creamy  mass,  made  up 
largely  of  pus  cells  with  cell  fragments  and  granular  detri- 
tus intermingled  with  large  and  smaller  clusters  of  readily 
stained  and  well-formed  tubercle  bacilli.  While  the  nuclei 
of  some  of  the  pus  cells  were  not  readily  stained  by  hema- 
toxylin, there  seemed  to  be  no  evidence  of  well-marked  co- 
agulation necrosis  in  the  cells  except  when  very  large 
masses  of  the  dead  bacilli  were  introduced.  This  central 
pus  mass  was  in  all  cases  closely  surrounded  and  inclosed 
by  a  wall  of  fibrous  tissue,  in  the  inner  layer  of  which  were 
numerous  larger  and  smaller  masses  of  epithelial  cells  in- 
termingled with  giant  cells.     Some  of  these  epithelial  and 


THE  ACTION  OF  DEAD  BACTERIA.        25 

giant  cell  islets  were  irregular  in  shape  and  diffuse  in  out- 
line. Others  were  sharply  circumscribed  and  rounded,  and 
presented  the  general  appearances  of  miliary  tubercles. 
Tubercle  bacilli  were  everywhere  abundant  in  these  epi- 
thelial cell  masses,  and  nowhere  else  in  the  fibrous  wall  of 
the  nodules.  Well-marked  cheesy  degeneration  was  no- 
where seen  in  these  cell  masses  which  resemble  tubercle  tis- 
sue, even  when  the  animal  remained  alive  for  four  weeks 
after  the  injection. 

We  thus  see  that  the  injection  of  dead  cultures  of  the 
tubercle  bacilli  is  capable  of  occasionally  inducing  in  the 
pleural  and  peritoneal  cavity  of  the  rabbit  a  suppurative  fo« 
cus  and  the  formation  about  it  of  a  tissue  morphologically 
similar  to  tubercle  tissue. 

While  these  results  are  interesting,  we  should  not  for- 
get that  a  great  variety  of  substances,  organized  and  unorgan- 
ized, on  introduction  into  the  serous  cavities,  as  well  as  be- 
neath the  skin,  are  capable  of  stimulating  the  cells  of  the 
part  to  the  production  of  a  new  tissue  which  in  many  re- 
spects closely  resembles  in  its  morphological  characters  the 
tissue  induced  by  living  tubercle  bacilli.  The  new  tissue 
growths  formed  under  these  conditions  have  been  the  sub- 
ject of  the  most  extended  research  in  times  gone  by.  And 
every  pathologist  who  lived  through  the  epoch  immediately 
preceding  the  discovery  of  the  tubercle  bacillus  knows  to 
what  fallacious  conclusions  experimental  studies  on  the  in- 
troduction of  foreign  bodies  into  animals  led  many  an  ob- 
server, owing  to  the  unwarrantable  stress  which  was  laid 
upon  epithelioid  cell  tissue  and  giant  cells,  in  determining 
the  morphology  of  tubercle. 

We  have  therefore  left  this  line  of  experiment  here  and 
turned  our  attention  to  a  form  of  experiment  which  seemed 
to  promise  a  more  direct  insight  into  the  detailed  effects  of 
dead  tubercle  bacilli  upon  living  animal  cells.  This  was  the 
injection  of  the  dead  germs  directly  into  the  blood-vessels 
of  the  rabbit  through  the  ear  veins. 

The  leading  idea  in  these  experiments  was  to  introduce 
into  the  body  the  dead  bacilli  under  such  conditions  that 
their  effects  upon  individual  cells  could,  if  possible,  be  de- 
termined. 

Intravenous  Injections. — We  have  used  in  these  experi- 


26  THE   ACTIOX   OF   DEAD   BACTERIA. 

ments  the  sterile  emulsions  of  tubercle  bacilli  described 
above,  and  have  injected  them  in  moderate  quantity,  aim- 
ing to  introduce  into  each  animal  an  amount  of  the  material 
which  would  correspond  to  a  spheroidal  mass  of  the  solid 
culture  of  from  about  one  to  two  millimetres  in  diameter, 
diffused  in  about  two  cubic  centimetres  of  water.  In  order 
to  avoid  gross  vascular  disturbance,  it  is  important  to  break 
up  as  much  as  is  practicable  the  flocculent  massesof  the  tu- 
bercle bacilli  with  a  platinum  needle  before  injecting  them. 
The  animals  bear  this  injection  into  the  blood  perfectly  Veil, 
and  we  have  in  our  series  of  experiments  in  this  way  rarely 
observed  that  their  general  health  was  in  any  way  impaired 
up  to  the  third  week,  when  a  small  proportion  of  the  ani- 
mals have  died  emaciated  (five  out  of  twenty-four).  The 
animals  were  killed  at  intervals  from  the  first  day  to  the 
second  month,  as  follows:  1st,  2d,  5th,  8th,  10th,  l7th, 
18th,  21st,  22d,  25th,  26th,  2'7th,  28th,  32d,  34th,  35th, 
48th,  60th  day.  The  organs  carefully  hardened  in  strong 
alcohol,  the  lungs  being  filled  through  the  trachea.  Some 
of  the  sections  were  stained  in  the  usual  way  with  hema- 
toxylin and  eosin,  while  others  were  stained  for  tubercle 
bacilli.  We  have  experimented  in  this  way  upon  twenty- 
four  rabbits. 

We  deem  it  best  to  give  a  summarized  statement  of  our 
results,  rather  than  a  detailed  account  of  the  individual  ex- 
periments, because  the  effects  of  the  dead  bacilli  vary  con- 
siderably, depending  upon  the  amount  introduced  and  the 
size  and  vigor  of  the  animal,  and  in  many  other  less  well- 
defined  ways. 

If  the  animal  is  killed  within  twenty-four  hours  after 
the  injection  of  a  moderate  amount  of  dead  tubercle  bacilli, 
the  organs  hardened  in  alcohol,  and  sections  finally  stained 
for  tubercle  bacilli,  it  will  be  found  that  the  bacilli  are 
present  in  by  far  the  largest  numbers,  and  in  the  largest 
masses  in  the  capillaries  of  the  lungs.  They  are  next  most 
abundant  in  the  capillaries  of  the  liver,  and  may  be  found 
in  small  numbers  in  the  spleen. 

After  the  first  three  or  four  days  we  have  been  unable 
to  find  bacilli  in  the  spleen,  but  they  appear  to  continue  in 
approximately  the  same  numbers  in  the  lungs  and  liver  as 
at   first.      We   have  made  no  examinations  of  the  other 


THE   ACTION   OF   DEAD   BACTERIA.  27 

viscera  with  a  view  of  determining  the  presence  or  distri- 
bution of  the  bacilli  in  them. 

We  need  only  refer  here  to  the  experiments  of  Wysso- 
kowicz  (12)  and  others  on  the  rapid  disappearance  of 
micro-organisms  injected  into  the  blood.* 

Examinations  of  sections  of  the  organs  of  animal$  killed 
during  the  first  and  second  week  after  the  injection  usually 
show  that  while  the  bacilli  have  largely  disappeared  from 
all  the  viscera,  except  the  lungs  and  liver,  they  may  still  be 
found  scattered  singly  or  in  small  clusters  in  the  smaller 
blood-vessels  of  these  organs.  Their  situation  can  usually 
be  best  made  out  in  the  capillaries  of  the  liver.  Here  they 
may  be  lying  apparently  free  against  the  wall  of  the  vessel, 
or  they  seem  often  to  be  surrounded  by  a  small  quantity  of 
a  nearly  homogeneous  or  finely  granular  material  which  may 
be  fibrin  or  blood  plates,  or,  finally,  they  may  lie  singly  or 
in  small  masses  inclosed  in  variously  shaped  cells. 

The  earliest,  and  throughout  the  experiments  the  most 
constant,  lesion  which  we  have  found  as  the  result  of  the 
venous  injection  of  dead  tubercle  bacilli  is  the  development 
in  the  lungs  of  from  one  or  two  to  innumerable  small  white 
nodules,  some  invisible  to  the  naked  eye,  some  as  large  as 
two  or  three  millimetres  in  diameter.  These  small  nodules 
may  appear  as  early  as  the  fifth  day  after  the  injection,  and 
persist  up  to  the  end  of  the  second  month.  In  the  earliest 
days  they  consist  of  a  central  denser  mass  made  up  of  epi- 
thelioid and  giant  cells,  interspersed  with  and  often  sur- 
rounded by  masses  of  small  spheroidal  cells  resembling 
leucocytes. 

Readily  stained  tubercle  bacilli  are  present  often  in  large 
numbers  in  these  lung  nodules,  especially  in  and  among  the 
epithelioid  and  giant  cells.  Such  a  nodule  with  its  bacilli 
stained  is  represented  in  Fig.  1. 

A  limited  area  of  lung  tissue  is  replaced  by  these 
nodules,  and  the  air-vesicles  about  them  show  an  increase  in 
the  epitheliod  cells,  which  sometimes  completely  fill  them. 

*  The  use  of  masses  of  dead  tubercle  bacilli  in  the  study  of  multiple 
embolism  would  suggest  itself  as  a  mode  of  experimentation  on  this 
subject,  since  the  differential  staining  enables  us  to  locate  the  seat  of 
lodgment  of  the  emboli  with  much  greater  accuracy  than  is  possible 
when  small  quantities  of  ordinary  insoluble  pigments  are  used. 


28  THE   ACTION   OF   DEAD   BACTERIA. 

In  animals  killed  at  a  later  period  these  nodules  are,  as 
a  rule,  denser  in  texture,  and  consist  largely  of  epithelioid 
cells  and  loose  fibrous  tissue  (Fig.  2).  Tubercle  bacilli  are 
now,  as  a  rule,  present  in  very  small  numbers. 

These  scattered  nodules  in  the  lungs  are  the  only  lesions 
which  ,we  have  found  up  to  the  third  week  ;  no  changes 
whatsoever  having  been  found  in  any  of  the  other  viscera 
before  this  time. 

Animals  killed  in  from  three  to  five  weeks  almost  in- 
variably show  a  considerable  number,  often  a  very  large 
number,  of  the  above-described  white  nodules  in  the  lungs, 
so  that  they  look  like  lungs  in  acute  miliary  tuberculosis- 
These  nodules  of  new-formed  tissue  are  usually  much  more 
easily  seen  after  the  lungs  have  been  distended  with  alco- 
hol and  hardened  than  before.  Tn  many  cases  animals 
show  no  other  gross  lesions  whatsoever,  either  in  the  kid- 
ney, spleen,  or  liver. 

But  if  a  microscopical  examination  of  the  liver  is  made, 
it  will  be  found  that  in  a  considerable  proportion  of  cases, 
as  early  as  the  third  week  after  the  injection,  swollen  en- 
dothelial cells  are  found  in  many  of  the  capillaries,  and 
scattered,  often  very  numerous,  islets  of  new-formed  cells 
ramify  in  spreading  clusters  in  the  capillaries  or  form  very 
minute  circumscribed  masses,  often  consisting  of  but  three 
or  four  cells  with  lai'ge  nuclei  and  faintly  granular  bodies 
(Fig.  3).  Sections  showing  these  lesions  and  stained  for 
tubercle  bacilli  show  almost  invariably  one  or  more  bacilli 
in  these  tiny  new-formed  cell  masses.  But  free  bacilli  or 
bacilli  inclosed  in  single  cells  are  rarely  to  be  found  in  the 
vessels,  as  they  are  in  the  first  fortnight  after  the  in- 
jections. 

In  most  of  the  animals  killed  in  from  four  to  six  weeks, 
in  addition  to  the  above  described  nodules  in  the  lungs,  the 
liver  was  more  or  less  thickly  bestrewn  with  minute  whit- 
ish spots  having  the  gross  appearance  of  miliary  tubercles. 
Microscopical  examination  of  these  livers  shows  that,  in  ad- 
dition to  these  macroscopical  lesions,  there  are  microscopical 
lesions  of  similar  character. 

The  simplest  alteration  that  is  found  at  these  later  pe- 
riods consists  in  a  proliferation  of  cells  within  the  capillaries 
of  the  liver,  so  that  these  are  moderately  and  diffusely  di- 


THE  ACTION  OF  DEAD  BACTERIA.        £9 

lated  in  places,  or  distended,  so  as  to  contain  branching 
nodules  of  epithelioid  cells  (see  Fig,  4).  It  seems  to  us  that 
these  new  cells  in  the  capillaries  are  in  part  at  least  derived 
from  the  vascular  endothelium  (see  Fig.  5). 

Sometimes  associated  with  this  diffuse  growth  of  new 
cells  in  the  capillaries,  sometimes  without  it,  the  whole  liver 
is  thickly  sprinkled  with  minute,  sharply  circumscribed, 
rounded  nodules.  Some  of  these  nodules  consist  of  epithe- 
lioid cells  in  part  sharply  outlined,  but  in  pait  merging  into 
one  another  and  intermingled  with  or  surrounded  by  a  zone 
of  small  spheroidal  cells  (Fig.  6). 

Many  of  the  small  nodules,  on  the  other  hand,  consist 
almost  exclusively  of  small  masses  of  epithelioid  cells,  either 
merged  together  as  in  Fig.  7,  or  loosely  packed  and  distinct 
as  in  Fig.  8.-  Again,  the  nodules  sometimes  consist  of 
a  single  small  mass  of  epithelioid  and  giant  cells,  as  in 
Fig.  9. 

Finally,  the  liver  nodules  may  be  more  complex  in  struct- 
ure and  consist  of  a  cluster  of  larger  and  sinaller  epithelioid 
and  giant-cell  masses,  as  in  Fig,  10.  We  have  seen  no  evi- 
dence of  proliferative  changes  in  the  liver  cells- ;  but  these 
are  sometimes  squeezed  and  flattened  by  the  growing  nod- 
ules. So  numerous  are  these  small  foci  of  cell  proliferation 
that  not  infrequently  from  two  to  ten  of  them  may  be  found 
within  the  area  of  a  section  of  a  single  liver  acinus.  They 
are  usually  situated  within  the  acinus,  more  rarely  in  the 
interlobular  connective  tissue. 

In  a  large  proportion  of  these  liver  nodules  a  varying 
number  of  tubercle  bacilli  can  be  found  (Figs.  3,  7,  9,  10). 
But  these  become  less  and  less  abundant  as  time  goes  on 
after  the  injection,  so  that  after  from  a  month  to  six  weeks 
we  have  found  but  a  few  scattered  bacilli  in  most  of  the 
nodules,  and  in  a  considerable  proportion  none  at  all.  The 
bacilli  do  not  appear  to  bear  any  special  relation  to  the 
giant  cells,  as  they  so  often  do  in  genuine  miliary  tubercles. 
They  are  often  considerably  broken  and  granular. 

It  is  most  noteworthy  in  this  connection  that  the  tuber- 
cle bacilli  are  much  more  abundant,  as  a  rule,  before  and 
during  the  early  stages  of  the  growth  of  these  cell  masses 
than  they  are  when  these  have  assumed  the  more  distinctly 
tubercular  type.     A¥e  have  found  no  lesions  whatsoever  in 


30  THE   ACTION   OF   DEAD    BACTERIA. 

any  of  the   animals,  in   the  kidneys,  and  the  tubercle-like 
masses  but  rarely  in  the  spleen. 

Oar  observations  on  the  later  stages  of  these  new  tissue 
growths  are  not  yet  complete  enough  to  enable  us  to  state 
what  becomes  of  them  when  the  animals  are  allowed  to  live. 
Up  to  the  end  of  the  seventh  week  we  have  seen  no  evi- 
dence of  cheesy  degeneration  even  in  the  largest  and  most 
complex  of  the  nodules.  It  is  perhaps  owing  to  the  lack  of 
cheesy  degeneration  that  the  tubercle-like  bodies  are  often 
first  revealed  by  the  microscopical  examination. 

We  do  not  mean  to  draw  fast  lines  in  assigning  definite 
periods  at  which  these  various  lesions  occur  after  injection 
of  the  dead  bacilli,  bnt  only  to  indicate  in  a  general  way 
the  averages  of  our  experiments. 

We  have  never  seen  the  slightest  indication  of  the  pro- 
liferation of  the  tubercle  bacilli  in  the  tissues  of  any  of  the 
injected  animals. 

The  introduction  of  the  dead  tubercle  bacilli  in  larger 
quantity  and  in  as  large  masses  as  can  be  used  without 
killing  the  animals  by  multiple  embolism  leads  to  some 
strikingly  different  results  from  those  which  we  have  de- 
scribed ;  but  this  series  of  experiments  is  not  yet  com- 
pleted and  need  not  be  further  considered  here. 

Summary. — We  have  found  that  prolonged  boiling, 
while  often  causing  a  considerable  breaking  up  of  the  tu- 
bercle bacillus,  does  not  interfere  with  its  characteristic 
staining  and  does  not  alter  the  morpliology  of  many  of  the 
individuals  of  a  culture ;  so  that  bacilli  killed  in  this  way 
and  introduced  into  the  body  can  be  readily  recognized 
at  their  seat  of  lodgment,  even  after  the  lapse  of  many 
weeks. 

Oiir  experiments  have  shown  that  dead  tubercle  bacilli 
separated  from  such  of  their  metabolic  products  as  are  set 
free  in  the  culture  media  or  are  extracted  by  prolonged 
bailing  in  water  or  fifty-per-cent.  glycerin  are  capable  of 
inducing  marked  effects  upon  the  body  cells  of  the  rabbit 
with  which  they  are  brought  in  contact. 

These  dead  tubercle  bacilli  are  markedly  chemotactic. 
When  introduced  in  considerable  amount  into  the  sub- 
cutaneous tissue  or  into  the  pleural  or  abdominal  cavities, 
they  are  distinctly  pyogenic,  causing  aseptic  localized  sup- 


THE   ACTION   OF   DEAD   BACTERIA.  31 

puration.  Under  these  conditions  they  are  capable,  more- 
over, of  stimulating  the  tissues  about  the  suppurative  foci 
to  the  development  of  a  new  tissue  closely  resembling  the 
diffuse  tubercle  tissue  induced  by  the  living  germs. 

We  have  found  that  dead  tubercle  bacilli  introduced  in 
small  numbers  into  the  blood-vessels  of  the  rabbit  largely 
disappear  within  a  few  hours  or  days,  but  that  scattering 
individuals  and  clusters  may  remain  here  and  there  in  the 
lungs  and  liver,  clinging  to  the  vessel  walls  for  many  days 
without  inducing  any  marked  changes  in  the  latter.  After 
a  time,  however,  earliest  in  the  lung ;  later,  as  a  rule,  in  the 
liver;  a  cell  proliferation  occurs  in  the  vicinity  of  these 
dead  germs  which  leads  to  the  formation  of  new  multiple 
nodular  structures  bearing  a  striking  morphological  resem- 
blance to  miliary  tubercles.  There  is  in  them,  however,  no 
tendency  to  cheesy  degeneration  and  no  evidence  of  pro- 
liferation of  the  bacilli,  but  rather  a  steady  diminution  in 
their  number.  It  seems  to  us  that  the  new  structures  origi- 
nate in  a  proliferation  of  the  vascular  endothelium  under 
the  stimulus  of  the  dead  and  disintegrating  germs. 

Control  Experiments. — It  will  no  doubt  appear  to 
others,  as  it  did  at  first  to  ourselves,  as  if  there  must  have 
been  some  experimental  error  in  the  attempts  to  kill  the 
bacilli,  so  singular  are  the  results  that  we  have  obtained. 
But  we  think  that  the  treatment  to  which  the  bacilli  used  in 
these  experiments  have  been  subjected,  together  with  the 
tests  of  death  to  which  we  have  exposed  them,  make  it  evi- 
dent that  we  have  actually  used  dead  germs  for  our  injec- 
tions. , 

This  would  appear  certain,  not  only  from  the  tests  which 
we  have  detailed  above,  but  also  from  the  fact  that  we  have 
made  fresh  boilings  for  each  set  of  experiments  (twelve  in 
number),  and  in  most  cases  have  prolonged  the  boiling  for 
from  one  to  three  hours  beyond  the  period  which  has  been 
established  as  fatal  to  these  germs.  It  should  be  remem- 
bered that,  according  to  the  researches  of  Schill  and 
Fischer  (13),  the  action  of  moist  heat  at  100°  C.  for  fifteen 
minutes  suffices  to  kill  tubercle  bacilli. 

The  question  now  arises  whether  the?e  curious  effects 
of  dead  tubercle  bacilli  upon  the  living  tissues  of  the  rabbit 
are  due  to  some  special  peculiarity  of  the  bodies  of  these 


32  THE   ACTIOX   OF   DEAD   BACTERIA. 

particular  germs,  or  may  be  induced  by  other  dead  germs 
or  other  substances  introduced  in  the  same  way. 

To  satisfy  ourselves  on  this  point,  we  have  made  a  series 
of  intravenous  injections  in  approximately  similar  amounts 
of  boiled  cultures  of  the  Bacillus  diphtherice  of  Loeffler  (six 
animals) ;  of  the  Bacillus  coli  communis  (five  animals)  ;  of 
tbe  Staphylococcus  pyogenes  aureus  (six  animals)  ;  of  wheat 
flour  (two  animals) ;  and  of  red  pepper  (two  animals). 
These  substances  have  been  introduced  aseptically.  The 
details  of  these  control  experiments  need  not  be  given  here, 
since  in  none  of  them  were  there  any  alterations  in  any 
way  comparable  to  those  so  nearly  uniformly  induced  by 
the  dead  tubercle  bacilli,  and  in  most  of  them  there  was  no 
reaction  or  change  at  all. 

The  animals  under  experiment  were  strictly  isolated  and 
kept  under  favorable  hygienic  conditions,  so  that  the  possi- 
bility of  a  chance  tuberculosis  is  largely  barred  out. 

We  must  therefore  conclude  that  the  lesions  caused  by 
the  introduction  of  dead  tubercle  bacilli  into  the  body  of 
rabbits  are  due  to  some  peculiar  property  of  the  bodies 
of  this  species  of  germ. 

Remarks. — Two  possibilities,  at  least,  exist  as  to  this 
peculiar  power  of  these  dead  tubercle  bacilli.  It  may,  of 
course,  possibly  be  due  to  some  trace  of  poison  elaborated 
in  the  germ  cell  during  its  life  and  not  given  up  in  the 
culture  medium,  or  extracted  by  boiling  in  water  or  dilute 
glycerin.  Or,  wbich  seems  more  probable  to  us,  in  view  of 
the  peculiar  properties  which  the  bacterio-protein  of  other 
species  of  germs  has  been  recently  shown  to  possess,  the 
remarkable  power  of  these  dead  germs  may  be  due  to  the 
specific  proteid  of  the  germ  cells,  which  is  gradually  set 
free  as  the  germs  disintegrate  at  their  seat  of  lodgment  in 
the  tissues. 

Though  we  have  made  repeated  attempts  to  isolate  the 
bacterio-protein  from  the  tubercle  bacilli  by  the  method  of 
Necki,  neither  with  this  nor  with  any  other  method  have 
we  succeeded. 

It  seems  to  us  highly  probable  that  the  bacterio- 
protein  of  the  tubercle  bacillus,  either  living  or  dead,  when 
set  free  by  disintegration  in  the  tissues,  is  capable  of  fur- 
nishing that  local  cell  stimulus  which  results  in  the  forma- 


THE   ACTION   OF   DEAD   BACTERIA.  33 

tion  of  the  various  phases  of  those  structures  whichi  we  call 
tubercles  aud  tubercle  tissue.  The  slow  growth  of  these 
structures  would  correspond  to  the  exceptional  structural 
invulnerability  of  the  tubercle  bacillus. 

These  structures,  which,  as  we  have  shown,  we  can  in- 
duce at  will  in  the  rabbit  by  the  injection  of  dead  tubercle 
bacilli  into  the  blood-vessels,  are  morphologically  absolutely 
typical  of  certain  phases  of  miliary  tubercles,  even  to  the 
presence  of  stainable  tubercle  bacilli  in  them.  Miliary  tu- 
bercles these  structures  are,  but — and  this  is  of  vital  impor- 
tance— the  animals  do  not  acquire  tuberculosis.  It  is  not 
an  acute  infectious  disease  which  we  thus  impart ;  there  are 
no  living  germs  to  grow.  The  disease,  if  it  is  a  disease, 
is  not  indefinitely  progressive.  The  dead  bacilli  seem  to 
act  as  foreign  bodies  simply,  curiously  stimulating,  it  is 
true,  but  only  dead  foreign  bodies  after  all. 

There  is  no  tendency  that  we  can  observe  in  these  tiny 
nodules  to  cheesy  degeneration. 

So  far  as  we  can  see  at  present,  when  the  limited 
amount  of  stimulant  or  poison,  proteid  or  other,  which 
they  contained  when  we  put  them  into  the  animal,  is  used 
up,  the  active  process  is  over,  and  it  rests  with  the  recupera- 
tive powers  of  the  animal  to  make  what  disposal  they  can 
of  the  local  ravages  which  have  been  caused. 

The  drawings  of  the  liver  of  rabbits  injected  through 
the  veins  with  living  tubercle  bacilli  by  Yersin  (14)  show 
at  a  glance  the  essential  difference  between  the  genuine 
miliary  tubercle  of  tuberculous  rabbits  and  the  non-infec- 
tious aseptic  tubercles  of  our  experiments — namely,  the 
actively  proliferating  tubercle  bacilli  which  make  the  lesion 
in  the  first  case  a  progvessive  one,  and  which,  sooner  or 
later,  cause  the  death  of  the  animal. 

These  studies  would  suggest  the  possibility  that  the 
formative  tissue  changes  which  are  characteristic  of  tuber- 
culosis in  man  may  be  largely  due  to  the  action  of  the  bac- 
terio-protein  of  the  tubercle  bacillus  set  free  as  the  germs 
die  and  disintegrate  in  the  tissues,  while,  on  the  other 
hand,  the  equally  important  and  in  some  ways  more  char- 
acteristic action  of  the  germ — namely,  the  coagulation  ne- 
crosis, and  possibly  some  of  the  vascular  changes  and  other 
poisonous  effects — may  be  chiefly  induced  by  those  freed 


34        THE  ACTION  OF  DEAD  BACTERIA. 

metabolic  products  of  the  life  processes  of  the  germ  which 
the  experiments  of  Koch  have  recently  brought  to  light,  or 
to  others  as  yet  unknown. 

We  resist  the  temptation  to  elaborate  here  the  many 
fascinating  possibilities  which  these  studies  suggest  as  to 
the  rationale  of  tubercular  inflammation  in  man,  and  the 
closer  analysis  of  its  lesions  which  may  be  possible  in  the 
future. 

If  the  results  of  our  experiments  are  confirmed  by 
others,  it  is  evident  that  our  conception  of  the  significance 
of  the  various  lesions  of  tuberculosis  may  be  considerably 
modified  and  the  therapeutic  possibilities  made  essentially 
clearer.  It  will  be  quite  comprehensible  why  so  many  well- 
defined  miliary  tubercles,  so  many  masses  of  tubercle  tis- 
sue, so  many  destructive  lesions  of  chronic  phthisis,  and  so 
many  so-called  scrofulous  and  tubercular  lymph  nodes  fail 
to  show  the  presence  of  tubercle  bacilli  if  it  is  established 
that  the  morphological  characteristics  of  these  struct- 
ures are  dependent  upon  the  disintegration  of  the  bacilli 
which  once  found  lodgment  where  they  form. 

We  are  aware  of  the  danger  of  drawing  far-reaching 
inferences  from  a  single  series  of  experiments.  But  we 
think  that  we  are  fairly  justified,  from  the  experimental 
data  now  set  forth,  in  the  conjecture  that  we  may  have  here 
the  explanation  of  the  comparatively  slow  growth  of  tuber- 
cle and  tubercle  tissue  which  is  observed  under  a  variety 
of  conditions. 

One  may  conjecture,  too,  whether  a  certain  number  of 
the  miliary  tubercles  which  we  find  in  the  body  after  a  gen- 
eralization of  the  tubercular  process  from  an  old  tubercu- 
lar focus  may  not  actually  be  innocuous  growths,  or  at  least 
harmful  only  as  foreign  bodies  in  the  tissues  where  they 
develop. 

The  possibility  would,  furthermore,  suggest  itself  that 
those  dense  masses  of  fibrous  tissue  in  the  lungs  which  we 
usually  regard  as  evidence  of  healed  tuberculosis  may  actu- 
ally be  innocuous,  even  though  they  harbor  stainable 
tubercle  bacilli,  and  no  longer  capable  of  lighting  up  a 
fresh  infection,  as  is  commonly  supposed. 

One  may  legitimately  qiiery,  too,  whether  in  the  closel" 
analysis  of  the  tubercular  processes  in  the  future  we  may 


THE   ACTION  OF   DEAD   BACTERIA.  35 

not  find  that  the  formation  of  the  new  tissue,  the  develop- 
ment of  the  tubercles  themselves,  is,  after  all,  an  attempt 
at  a  conservative  process  on  the  part  of  the  organism  in- 
cited by  the  stimulus  of  the  disintegrating-  bodies  of  some 
of  the  germs.  It  may  be,  too,  that,  as  Hueppe  suggests, 
the  conservative  attempt  is  often  rendered  futile  by  a  de- 
structive tendency — cheesy  degeneration — developed  under 
the  influence  of  a  metabolic  product  of  the  living  and  grow- 
ing germs  which  the  tubercular  foci  still  harbor.  The 
bearing  of  this  possibility  upon  the  administration  of  large 
doses  of  Koch's  tuberculin  should  not,  perhaps,  be  wholly 
ignored  even  at  this  stage  of  our  knowledge. 

It  is  somewhat  curious  to  notice  how  closely  in  accord 
with  some  of  our  experimental  results  are  many  of  the  feat- 
ures of  Hueppe's  (15)  recent  essay  to  develop  a  rational 
explanation  on  theoretical  grounds  of  the  varied  phases  of 
the  development  of  tubercles  and  tuberculosis. 

We  wish,  in  conclusion,  to  call  renewed  attention  to 
the  fact,  too  often  overlooked,  that  the  acute  infectious  dis- 
eases are  much  more  complex,  both  in  their  lesions  and 
their  aetiology,  than  we  are  wont  to  realize.  We  were  for- 
merly perforce  content  when  we  had  done  what  we  could 
to  make  plain  the  morphology  of  their  obvious  lesions. 
Light  dawned  at  last  on  the  relationship  of  bacteria  to  some 
of  these  diseases,  and  the  definite  establishment  of  the  con- 
stant and  inevitable  relationship  between  some  specific  germ 
and  its  special  disease  was  at  first  so  satisfying  that  this 
relationship  alone  was  regarded  as  a  solution  of  its  fetiology. 

But  the  restless  seeker  after  detail  was  not  long  content 
with  this,  and  new  light  was  speedily  forthcoming  as  to 
the  important  role  which  chemical  poisons,  elaborated  by 
pathogenic  germs,  had  to  play  in  the  induction  of  the  varied 
phenomena  of  this  class  of  diseases.  We  have  seen  that  a 
new  phase  of  our  knowledge  is  being  now  developed  in 
the  study  of  the  proteid  ingredients  of  the  germ  bodies. 

But  beyond  all  these  lines  of  study  and  essential  to 
their  full  fruition  is  the  development  of  a  fuller  comprehen- 
sion of  the  powers  and  reactions  and  idiosyncrasies  of  the 
body-cells  themselves,  upon  whose  comportment  in  the  last 
analysis  depend  the  distinctive  features  and  the  ultimate 
siacnificance  of  the  acute  infectious  diseases. 


36  THE    ACTION   OF   DEAD   BACTERIA. 

Thus  the  harvest  garnered  in  this  newly  opened  field  of 
thought  and  research  must  ultimately  be  brought  home  to 
the  old  storehouse  of  pathological  anatomy  and  physiology 
before  it  can  be  made  in  the  fullest  degree  available  for  the 
understanding  and  curtaihnent  of  bacterial  disease. 

1.  Prudden.     New  York  MefUeal  Journal^  Jiine  6,  1891. 

2.  Haramerschlag.  Rev^Ctrlhl.f.  Bacteriologie,  etc.,  March 
2,  1891,  p.  272. 

3.  Hueppe  and  Scholl.  Berlin,  klin.  TF(?cAe?^^c7i?•.,  1891,  Jan. 
26,  Feb.  23,  March  16,  March  23. 

4.  Trudeau.     Medical  Record,  Nov.  22,  1890. 

5.  Zulzer.     Berlin.  Tclin.  Wochenschr.,  1890,  No.  4. 

6.  Crookshank.     Lancet,  Feb.  27,  1891,  p.  300. 

7.  Weyl.     Deutsch.  med.  Wochenschr.,  Feb.  12,  1891,  p.  256. 

8.  Maffacci.     CtrlU.f.  allg.  Path.,  etc.,  Dec.  15,  1890. 

9.  Wyssokowicz.  Mitth.a.  Dr.  Bremer''sHeilanstalt,u.s.w., 
neue  Folge,  1890. 

10.  Koch.    Reprint,  Medical  Record,  Jan.  17,  1891,  p.  85. 

11.  Hneppe  and  Scholl.  Berlin,  klin.  Woehenschrift,  Jan. 
26,  1891,  p.  89. 

12.  Wyssokowicz.     Zeitschrift fur  Hygiene,  Bd.  i,  p.  3. 

13.  Schill  and  Fischer.  Mitth.  a.  d.  kaiserl.  Oesund- 
heitsamte,  Bd.  ii. 

14.  Yersin.     Annales  de  VlnstitUt  Pasteur,  1888.  p.  140. 

15.  Hiieppe.     Berlin.  Min.  Wochenschr.,  M;trcli  23,  1891. 


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